The goal of this project is to assess the therapeutic potential of novel antifungal agents, identified by our laboratory via screening a ChemBridge library, that target the synthesis of fungal but not mammalian glucosylceramide (GlcCer). Recently, it has been reported by us and other investigators that fungal GlcCer is required for virulence of many fungi, including Cryptococcus neoformans (Cn),1,2 Candida albicans (Ca),3-5 Aspergillus fumigatus (Af)6 and others.7 In addition, GlcCer is detected only in the host infective form (yeast) and not in the environmental form (mold) of many dimorphic fungi.8-10 Furthermore, the synthesis of GlcCer seems to be important also for Pneumocystis pneumonia (PCP) as glucosylceramide synthase transcripts are highly elevated at the time of isolation of the fungus from a fulminate lung infection.11 Studies in our and other labs revealed that GlcCer promotes alkaline tolerance in fungi.6,12-14 Particularly, GlcCer regulates fungal cell replication by promoting cell cycle progression and cytokinesis in a neutral/alkaline but not acidic environment.6,12,14 Taken together, these studies suggest that GlcCer is most likely a pan-fungal virulence factor required during infection to promote fungal growth at neutral/alkaline environments (e.g. alveolar spaces and bloodstream), and as such, it is a promising novel drug target. Therefore, we looked for inhibitors of GlcCer synthesis by screening a ChemBridge library for compounds that inhibit fungal growth in an environment similar to the lung and bloodstream: neutral/alkaline pH, 37C and 5% CO2 using Cn as a model organism. We identified 2 compounds (BHBM and its derivative D0) that significantly decreased the synthesis of GlcCer in Cn but not in mammalian cells (Fig. 1).15 The compounds are fungicidal (Fig. 2) and able to improve mice survival during invasive cryptococcosis, candidiasis and pneumocystosis (Fig. 3).15 Mechanistic studies show that the compounds target SLA2, a gene controlling fungal vesicle trafficking (Fig. 6), which is how ceramide is transported for the synthesis of GlcCer. Therefore, we hypothesize that targeting the fungal GlcCer pathway will be an effective novel therapeutic strategy for impeding the development of fungal diseases. To test this hypothesis, we propose the following aims: Aim 1. Medicinal Chemistry. Employ combinatorial/parallel synthesis to create a first-generation library of fungal GlcCer inhibitors based on early lead compounds BHBM and D0. Obtain ca. 300 compounds for initial screening for solubility, antifungal activity, toxicity studies and generate an optimization library of ca. 30 compounds to identify a dozen advanced lead compounds, all possessing good in vitro antifungal activity and with a EC50 cytotoxicity/MIC80 efficacy ratio >100 (based on studies in Aim 2). These compounds will then be tested in vivo for pharmacokinetics (PK) and animal studies (Aim 3). Aim 2. Mechanism of action and resistance development. Confirm and validate the compounds synthesized in Aim 1 for ability to inhibit fungal but not mammalian GlcCer/SLA2. Determine whether cells can develop resistance to the candidate compounds. Iterate library based on observed Structure-Activity-Relationship (SAR). Aim 3. PK and in vivo toxicity and efficacy. Study PK of lead compounds selected from Aim 2 and assesses their effect in in vivo models of cryptococcosis, candidiasis, pneumocystosis and aspergillosis.